The present invention relates to compositions related to proteins which function in controlling physiology, development, and/or differentiation of mammalian cells. In particular, it provides proteins which are implicated in the regulation of physiology, development, differentiation, or function of various cell types, e.g., chemokines, 7 transmembrane receptors, reagents related to each, e.g., antibodies or nucleic acids encoding them, and uses thereof.
The circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematoloqy, Little, Brown and Co., Boston, Mass.; and Paul (ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.
For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the xe2x80x9cimmune network.xe2x80x9d Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which should lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system and other disorders.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of the pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
The chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the xe2x80x9cCxe2x80x94Cxe2x80x9d branch), or spaced by an intervening residue (xe2x80x9cC-X-Cxe2x80x9d). A more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins. Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy and Immunol. 109:97-109.
The chemokine receptors are typically members of the superfamily of G-protein coupled (or linked) receptors (GPCR, or GPLR). As a class, these receptors are integral membrane proteins characterized by amino acid sequences which contain seven hydrophobic domains. See, e.g., Ruffolo and Hollinger (eds. 1995) G-Protein Coupled Transmembrane Signaling Mechanisms CRC Press, Boca Raton, Fla.; Watson and Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic Press, San Diego, Calif.; Peroutka (ed. 1994) G Protein-Coupled Receptors CRC Press, Boca Raton, Fla.; Houslay and Milligan (1990) G-Proteins as Mediators of Cellular Signaling Processes Wiley and Sons, New York, N.Y.; and Dohlman, et al. (1991) Ann. Rev. Biochem. 60:653-688. These hydrophobic domains are predicted to represent transmembrane spanning regions of the proteins. These GPCRs are found in a wide range of organisms and are typically involved in the transmission of signals to the interior of the cell, e.g., through interaction, e.g., with heterotrimeric G-proteins. They respond to a wide and diverse range of agents including lipid analogs, amino acid derivatives, small peptides, and other molecules.
The presumed transmembrane segments are typically 20-25 amino acids in length. Based upon models and data on bacteriorhodopsin, these regions are predicted to be a-helices and be oriented to form a ligand binding pocket. See, e.g., Findley, et al. (1990) Trends Pharmacol. Sci. 11:492-499. Other data suggest that the amino termini of the proteins are extracellular, and the carboxy termini are intracellular. See, e.g., Lodish, et al. (1995) Molecular Cell Biology 3d ed., Scientific American, New York; and Watson and Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic Press, San Diego, Calif. Phosphorylation cascades have been implicated in the signal transduction pathway of these receptors.
Although the full spectrum of biological activities mediated by these 7 transmembrane receptors has not been fully determined, chemoattractant effects are recognized. Chemokine receptors are notable members of the GPCR family. See, e.g., Samson, et al. (1996) Biochemistry 35:3362-3367; and Rapport, et al. (1996) J. Leukocyte Biology 59:18-23. The best known biological functions of these chemokine molecules relate to chemoattraction of leukocytes. However, new chemokines and receptors are being discovered, and their biological effects on the various cells responsible for immunological responses are topics of continued study.
Many factors have been identified which influence the differentiation process of precursor cells, or regulate the physiology or migration properties of specific cell types. These observations indicate that other factors exist whose functions in immune function were heretofore unrecognized. These factors provide for biological activities whose spectra of effects may be distinct from known differentiation or activation factors. The absence of knowledge about the structural, biological, and physiological properties of the regulatory factors which regulate cell physiology in vivo prevents the modulation of the effects of such factors.
In addition, other factors exist whose functions in hematopoiesis, neural function, immune development, and leukocyte trafficking were heretofore unrecognized. These receptors mediate biological activities whose spectra of effects are distinct from known differentiation, activation, or other signaling factors. The absence of knowledge about the structural, biological, and physiological properties of the receptors which regulate cell physiology, development, or function prevents the modification of the effects of such factors.
Thus, medical conditions where regulation of the development or physiology of relevant cells is required remain unmanageable.
The present invention is based, in part, upon the discovery of new genes encoding various chemokines, e.g., those designated CXC N4; or 7 transmembrane receptors, e.g., those designated DNAXCCR10, which encode rodent receptors; and BLRx, which encode primate receptors. Each GPCR gene encodes a polypeptide exhibiting structural and/or sequence homology to 7 transmembrane receptors. Such receptors are typically G-protein coupled (or linked) receptors (GPCR or GPLR), though the complete set of ligands for each has not yet been identified.
The invention also provides mutations (muteins) of the respective natural sequences, fusion proteins, chemical mimetics, antibodies, and other structural or functional analogs. It is also directed to isolated nucleic acids, e.g., genes encoding respective proteins of the invention. Various uses of these different protein, antibody, or nucleic acid compositions are also provided.
The present invention provides a composition selected from the group of: a substantially pure antigenic polypeptide comprising sequence from a CXC N4, a DNAXCCR10, or BLRx; a binding composition comprising an antigen binding portion of an antibody specific for binding to such an antigenic polypeptide; a nucleic acid encoding such an antigenic polypeptide; and a fusion protein comprising at least two non-overlapping segments of at least 10 amino acids of such an antigenic polypeptide.
In certain embodiments of the antigenic polypeptide, it is from a warm blooded animal, e.g., a rodent or primate; it comprises a sequence of FIGS. 1-5; it exhibits a post-translational modification pattern distinct from a natural form of said polypeptide; it is detectably labeled; or it is made by expression of a recombinant nucleic acid. In other embodiments, a sterile form is provided, including, e.g., composition comprising the polypeptide and an acceptable carrier. A detection kit comprising a compartment or container holding such an antigenic polypeptide is also provided.
In other binding composition forms, e.g., antibody embodiments, the polypeptide is a mouse or human protein; the antibody is raised against a peptide sequence of FIGS. 1-5; the antibody is a monoclonal antibody; the binding composition is fused to a heterologous protein, or is detectably labeled. An alternative embodiment is a binding compound comprising an antigen binding fragment of the antibody described. Also provided is a detection kit comprising such a binding compound. With the antibodies are provided methods of purifying a polypeptide using the binding compound or antibody to specifically separate the polypeptides from others, or for detection, e.g., immunohistochemistry or immunoprecipitation.
Nucleic acid embodiments are provided, e.g., where the nucleic acid is in an expression vector and: encodes a polypeptide from a mouse or human; comprises a sequence of a mature protein of FIGS. 1-5; or comprises a deoxyribonucleic acid nucleotide. The invention also provides a kit with such nucleic acids, e.g., which include PCR primers for amplifying such sequences.
With nucleic acids are provided fusion proteins, comprising: a sequence of FIGS. 1-5; and/or sequence of another chemokine or 7 transmembrane receptor, as appropriate. Also provided is a cell comprising a recombinant nucleic acid, as described, and methods of producing a polypeptide comprising expressing the nucleic acid in an expression system.
Other embodiments include methods of modulating physiology or development of a cell, or treating a disorder, with a step of contacting that cell with a composition comprising an agonist or antagonist of the chemokine or receptor. Ordinarily, the cell is a neuron, macrophage, a lymphocyte, or a skin cell, such as found in the epidermis or dermis. Various physiological effects to be modulated include a cellular calcium flux, a chemoattractant response, cellular morphology modification responses, phosphoinositide lipid turnover, an antiviral response, or a proliferative response.
Yet further embodiments are directed to a method comprising administering to a subject in need thereof an effective amount of an agent that modulates the expression or activity of BLRx or fragments thereof. In a further embodiment, the method of treatment or prevention comprises administering to a subject in need thereof an effective amount of an agent that enhances or decreases the activity or expression of BLRx or fragments thereof. The agent can be, for example, a polynucleotide encoding BLRx, a polynucleotide that includes a transcriptional or translational regulator, an antibody that specifically binds to BLRx, an antisense oligonucleotide having a sequence that binds to a sequence encoding BLRx, or a small molecule inhibitor.
The present methods can be directed to the treatment of various disorders, including, for example, wound healing, proliferative disorders, fibrotic disorders, sclerotic disorders, cancer, and angiogenesis.
In another aspect, the present invention encompasses pharmaceutical compositions comprising an effective amount of an agent that modulates the expression or activity of BLRx and a suitable carrier. The agent can be a BLRx agonist or antagonist, for example, or can comprise BLRx and a suitable carrier.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.